A recently identified protein, FAN1 (FANCD2-associated nuclease 1, previously known as KIAA1018), is a novel nuclease associated with monoubiquitinated FANCD2 that is required for cellular resistance against DNA interstrand crosslinking (ICL) agents. The mechanisms of FAN1 regulation have not yet been explored. Here, we provide evidence that FAN1 is degraded during mitotic exit, suggesting that FAN1 may be a mitotic substrate of the anaphase-promoting cyclosome complex (APC/C). Indeed, Cdh1, but not Cdc20, was capable of regulating the protein level of FAN1 through the KEN box and the D-box. Moreover, the up- and down-regulation of FAN1 affected the progression to mitotic exit. Collectively, these data suggest that FAN1 may be a new mitotic substrate of APC/CCdh1 that plays a key role during mitotic exit.
Anaphase-Promoting Complex-Cyclosome ; Bone Neoplasms ; metabolism ; pathology ; Cadherins ; genetics ; metabolism ; Cdc20 Proteins ; Cell Cycle Proteins ; genetics ; metabolism ; Cell Line, Tumor ; Exodeoxyribonucleases ; genetics ; metabolism ; HEK293 Cells ; Humans ; Mitosis ; Osteosarcoma ; metabolism ; pathology ; Ubiquitin-Protein Ligase Complexes ; genetics ; metabolism

To express and identify fibronectin C-terminal heparin-binding domain (FNCH BD) polypeptides in Pichia pastoris expression system and study its function, the fragment of FNCHBD was amplified by PCR and inserted into pGEM-T vector. After sequenced, the fragment was inserted into pAo815SM vector, and then cloned into the expression vector pPIC9k. The recombinant plasmid was linerarized with restrict enzyme Sal I and transferred into the yeast host cell KM71 and GS115. The positive yeast clone was screened by G418 resistant, and the target protein was induced to express in the medium containing 0.5% methanol. The culture supernatant was collected and then was purified with membrane ultrafiltration and ion exchange chromatography. The purified product was analyzed with mass spectrogram, SDS-PAGE, Western blotting and heparin affinity chromatography. The results showed that the target protein was around 32 kDa and the purity of the product was above 95%. FNCHBD could be specifically recognized by fibronectin polyclonal antibody. These results suggest that FNCHBD could be expressed and purified successfully in Pichia pastoris, which provides a good strategy to further studies.
Fibronectins ; biosynthesis ; chemistry ; genetics ; Genetic Vectors ; Heparin ; metabolism ; Peptides ; genetics ; metabolism ; Pichia ; genetics ; metabolism ; Protein Binding ; Protein Structure, Tertiary ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics

OBJECTIVETo study the anticancer activities of curcumin on human Burkitt's lymphoma and their molecular mechanism.

METHODSThe effect of curcumin on the growth of CA46 cells and apoptosis were studied through Trypan blue exclusion, MTT assay, cell cycle, DNA fragmentation analysis and detection of TdT-mediated dUTP nick end labeling (TUNEL). The effect of curcumin on the expression of c-myc, bcl-2, mutant-type p53 and Fas protein and mRNA was studied by flow cytometry (FCM) and reverse transcription-polymerase chain reaction (RT-PCR).

RESULTS1. Curcumin inhibited proliferation of CA46 cells in a time- and dose-dependent manner, 2. CA46 cells treated with curcumin showed G(0)/G(1) or G(2)/M phase increase and S phase decrease, 3. CA46 cells apoptosis induced by curcumin was confirmed by DNA fragmentation and TUNEL and 4. The expression of c-myc, bcl-2, mutant-type p53 protein and mRNA was decreased sharply in CA46 cells treated with curcumin, while Fas protein and mRNA was increased.

CONCLUSIONCurcumin is able to inhibit the proliferation of CA46 cells and induce the cell apoptosis by down-regulating the expression of c-myc, bcl-2, mutant-type p53 and up-regulating the expression of Fas.

Antineoplastic Agents ; pharmacology ; Apoptosis ; Burkitt Lymphoma ; Cell Cycle ; drug effects ; Cell Division ; drug effects ; Curcumin ; pharmacology ; Gene Expression Regulation ; drug effects ; Humans ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; Proto-Oncogene Proteins c-myc ; biosynthesis ; genetics ; Tumor Cells, Cultured ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics ; fas Receptor ; biosynthesis ; genetics